Direct Evidence That the Reaction Intermediate of Metallo-β-lactamase L1 Is Metal Bound

In an effort to probe the structure of the reaction intermediate of metallo-β-lactamase L1 when reacted with nitrocefin and other β-lactams, time-dependent absorption and rapid-freeze-quench (RFQ) EPR spectra were obtained using the Co(II)-substituted form of the enzyme. When using nitrocefin as the substrate, time-dependent absorption spectra demonstrate that Co(II)-substituted L1 utilizes a reaction mechanism, similar to that of the native Zn(II) enzyme, in which a short-lived intermediate forms. RFQ-EPR spectra of this intermediate demonstrate that the binding of substrate results in a change in the electronic properties of one or both of the Co(II)\u27s in the enzyme that is consistent with a change in the coordination sphere of this metal ion. This observation provides evidence that the reaction intermediate is a metal-bound species. RFQ-EPR studies also demonstrate that other β-lactams, such as cephalothin, meropenem, and penicillin G, proceed through an electronically similar complex and that the role of metal is similar in all cases. EPR spectroscopy has also identified distinct product-bound species of L1, indicating that reversible product binding must be considered in all future kinetic mechanisms. Consideration of the time-dependent optical and EPR studies in light of available crystallographic information indicates the intimate involvement of the metal ion in the Zn2-binding site of L1 in the hydrolytic reaction

A continuously updated, geospatially rectified database of utility-scale wind turbines in the United States.

Over 60,000 utility-scale wind turbines are installed in the United States as of October, 2019, representing over 97 gigawatts of electric power capacity; US wind turbine installations continue to grow at a rapid pace. Yet, until April 2018, no publicly-available, regularly updated data source existed to describe those turbines and their locations. Under a cooperative research and development agreement, analysts from three organizations collaborated to develop and release the United States Wind Turbine Database (USWTDB) - a publicly available, continuously updated, spatially rectified data source of locations and attributes of utility-scale wind turbines in the United States. Technical specifications and wind facility data, incorporated from five sources, undergo rigorous quality control. The location of each turbine is visually verified using high-resolution aerial imagery. The quarterly-updated data are available in a variety of formats, including an interactive web application, comma-separated values (CSV), shapefile, and application programming interface (API). The data are used widely by academic researchers, engineers and developers from wind energy companies, government agencies, planners, educators, and the general public

Agroforestry and the achievement of the Millennium Development Goals.

Abstract The Millennium Development Goals (MDGs) of the United Nations (UN) are at the heart of the global development agenda. This chapter examines the role of agroforestry research and development (R&D) in light of the MDGs. It reviews some of the ways in which agroforestry is substantively assisting to achieve the goals and discusses how the agenda can be realigned to further increase its effectiveness in helping developing countries to meet their MDG targets. Promising agroforestry pathways to increase on-farm food production and income contribute to the first MDG, which aims to cut the number of hungry and desperately poor by at least half by 2015. Such pathways include fertilizer tree systems for smallholders with limited access to adequate crop nutrients, and expanded tree cropping and improved tree product processing and marketing. These advances can also help address lack of enterprise opportunities on small-scale farms, inequitable returns to small-scale farmers (especially women), child malnutrition, and national tree-product deficits (especially timber). The rate of return to investment in research on tree crops is quite high (88%); but enterprise development and enhancement of tree-product marketing has been badly neglected. The products, processing, and marketing of tree products and services, through tree domestication and the commercialization of their products is a new frontier for agroforestry R&D. A major role for agroforestry also is emerging in the domain of environmental services. This entails the development of mechanisms to reward the rural poor for the environmental services such as watershed protection and carbon sequestration that they provide to society. Agroforestry R&D is contributing to virtually all of the MDGs. But recognition for that role must be won by ensuring that more developing countries have national agroforestry strategies, and that agroforestry is a recognized part of their programs to achieve the MDGs

Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

BACKGROUND: The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. RESULTS: Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying K(m) and k(cat) values for the different enzymes and substrates and that no direct correlation between K(m) and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. CONCLUSIONS: The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that K(m) values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases